Protein detection and characterization is an important task for pharmaceutical and clinical research. Chemiluminescence (CL) is a common method for detection of proteins in biochemical analyses or on surface-bound and spatially separated proteins. An example of the latter is the method of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with electrophoretic transfer of proteins to a membrane, referred to as Western Blot (WB) analysis (Towbin et al. (1979) Proc. Natl. Acad. Sci. U.S.A. 76(9):4350-4354; Renart et al. (1979) Proc. Natl. Acad. Sci. U.S.A. 76(7):3116-3120). Electro-chemiluminescence (ECL) has also been applied to detect proteins bound to spots in specially designed multiwell plates (e.g., MULTI SPOT® and MULTI-ARRAY™ plates and SECTOR™ instruments, Meso Scale Discovery, a division of Meso Scale Diagnostics, LLC, Gaithersburg, Md.).
An advantage of CL and ECL is very high sensitivity with limits of detection for proteins in solution in the sub-picogram/ml range. However, these systems produce transient signals, are not chemically stable, and require a complicated procedure to produce the chemical reaction required for detection. They are also non-linear systems (i.e., one probe produces many photons) and have poor reproducibility so are not suitable for applications where quantitation of protein amount is desired. A last, but significant limitation is the inability to multiplex multiple CL signals. Their emissions are very broad and that makes the ability to detect two different CL emissions from the same spatial location very challenging.
Fluorescence (FL) probes overcome some of the limitations of CL. They provide ability for better quantitation since the relationship between excitation photons and emission photons is, in general, linear. They are also more versatile as there is no need to provide access to the probes by other reactive molecules. In general, FL probes are also more stable, especially when protected from light as they are generally non-reactive chemical species. Perhaps the most important advantage of FL probes is that they provide the ability to perform multiplexing. FL molecules come in a wide variety of forms with a wide range of excitation and emission bands. Thus two (or more) probes at the same spatial location can be independently excited and detected with minimal overlap (or cross-talk) between detection channels. The ability to detect up to four independent fluorophores from the same spatial location is regularly reported using color bandpass filters. Higher levels of multiplexing have been reported with flow cytometry and multispectral imaging (Stack et al. (2014) Methods 70(1):46-58; Perfetto et al. (2004) 4(8):648-655).
Unfortunately, FL probes have not demonstrated the same level of sensitivity as CL and typically have a lower dynamic range. A reason for lower sensitivity with FL probes is the presence of background from autofluorescence of co-localized material or interference of fluorescence from other probes. A different technique was developed to reduce background from autofluorescence using longer lifetime fluorescent probes called time-resolved fluorescence (TRF) (Zuchner et al. (2009) Anal. Chem. 81(22):9449-9453; Kemper et al. (2001) Electrophoresis. 22(5):881-889; Lim et al. (1997) Anal Biochem. 245(2):184-195; Huhtinen et al. (2005) Anal. Chem. 77(8):2643-2648; Vereb et al. (1998) Biophys J. 74(5):2210-2222). In brief, autofluorescence typically has a relatively short lifetime (<20 ns) so that TRF detection is delayed in time until after the autofluorescence signal has died away. This is technically time gated detection, but has commonly been called time-resolved (Lakowicz, “Principles of Fluorescence Spectroscopy,” 3rd Edition, Springer-Verlag, New York, 2006). The benefits of TRF detection have been well documented and include higher sensitivity, lower background, and wider dynamic range (Eliseeva & Bunzli (2010) Chem. Soc. Rev. 39(1):189-227; Bunzli & Piguet (2005) Chem. Soc. Rev. 34(12):1048-1077; Diamandis (1991) Clin. Chem. 37(9):1486-1491).
Multiplexing of TRF has been reported with some success. The use of Eu and Tb based probes has been demonstrated in biochemical assays using Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) to detect two different proteins (Degorce et al. (2009) Curr. Chem. Genomics. 3:22-32; Bookout et al. (2000) J. Agric. Food Chem. 48(12):5868-5873; Hamy et al. (2001) J. Biomol. Screen. 6(3):179-187). In addition, there have also been reports of multiplexing with Eu and Sm, and Eu, Tb, and Sm (Bador et al. (1987) Clin. Chem. 33(1):48-51; Heinonen et al. (1997) Clin. Chem. 43(7):1142-1150). However, these systems suffer from cross talk as emission from one of the lanthanides bleeds into the detection channels of the other lanthanides. This limits the utility of these methods to having only one truly sensitive channel, while the other is limited by background signal from the second species.
Therefore, there is a need for an improved multiplexed system that maintains the high sensitivity, background rejection, stability, resistance to photo bleaching, and dynamic range of time-resolved fluorescence detection with minimal or no cross talk between channels.